Intake and digestibility of four rations with different fiber levels in alpacas (Vicugna pacos).

The aim of this study was to evaluated the impact of different fibre levels in alpaca diet on voluntary feed intake and apparent digestibility, and to estimate the digestibility of organic matter (OMD) from the content of crude protein (CP) in feces. The study was carried out with twelve alpacas (36.7 ± 6.4 kg body weight- BW), which were offered 4 treatments with different neutral detergent fiber content (NDF. T1: 40.3%; T2: 62%; T3: 68%; T4: 72%) under a switch back design. Absolute daily dry matter intake (DMI) was higher for T1 (678 g/d) than T4 (312 g/d) (p ≥ 005). NDF intake was similar between treatments when related to BW or MW (on average 1% BW and 22 g/kg MW. p ≥ 0.05). Water intake (L/kg DMI) was higher in T1 compared to the other treatments, with values ranging from 2.9 L/kg DMI(T1) to 2.8 L/kg DMI(T4), respectively (p ≤ 0.05). Digestibility of dry matter, organic matter and CP was higher in T1 than in the other treatments, with average values ranging from 72% for T1 to 32% for T4 (p ≤ 0.05). NDF digestibility was similar among treatments (p ≥ 0.05). The regression equation generated to predict OMD (y) was as follows: y = 0.360 + 0.08294*fecal CP (g/kg OM). Further studies will indicate whether faecal nitrogen can be used to estimate digestibility and hence diet quality in South American camelids.

Activation of caspase-3/7, an apoptotic-related marker, during incubation and cryopreservation of alpaca (Vicugna pacos) spermatozoa.

Caspases are crucial mediators of programmed cell death (apoptosis). Apoptosis can occur in spermatozoa during spermatogenesis or epididymal transit, as well as in ejaculated spermatozoa. A high proportion of apoptotic sperm would be a poor indicator of the freezability of a raw seminal sample. Alpaca spermatozoa are notoriously difficult to freeze successfully. Therefore, the objectives of this study were to study caspase activation during incubation (37°C) of fresh alpaca spermatozoa, as well as before and after cryopreservation, to gain some insight into the mechanisms behind the vulnerability of alpaca spermatozoa. Eleven sperm samples were incubated for 4 h at 37°C (Study 1), and 23 samples were frozen using an automated system (Study 2). Caspase-3/7 activation was assessed at 0,1,2,3, and 4 h in samples incubated at 37°C (Study 1); and before/after cryopreservation (Study 2) using CellEvent™ Caspase 3/7 Green Detection Reagent and flow cytometry. The proportions of alpaca spermatozoa with caspase-3/7 activated increased (p < 0.05) after 3-4 h of incubation at 37°C; however, caspase activation was similar before and after cryopreservation (36.2 ± 11.2% vs. 36.6 ± 33.7%, p > 0.05). The high standard deviation found after freezing could be explained by the existence of two subpopulations: one subpopulation where caspase-3/7 activation decreased during cryopreservation (from 36.6 ± 9.1% to 1.5 ± 2.2%), and the other subpopulation where caspase-3/7 activation increased after cryopreservation (from 37.7 ± 13.0% to 64.3 ± 16.7%). In conclusion, after 3-4 h of incubation, caspase-3/7 activation increased in fresh alpaca sperm, whereas cryopreservation affects alpaca sperm samples in different ways.

The effect of photoperiod and melatonin on plasma prolactin concentrations in female guanaco (Lama guanicoe) in captivity.

The present study examined the effects of different photoperiods and melatonin treatment on plasma prolactin concentrations in guanacos, a South American camelid, in captivity. Fourteen adult female guanacos, not gestating or lactating and isolated from males, were studied. The control group was exposed to natural daylight, during short days (N = 7, 10L:14D) and long days (N = 7, 16L:8D). The treatment group (N = 7, 10L:14D) received melatonin implants every 23 days for 6 weeks during long days. Blood samples were taken at intervals of 1 week for 3 weeks, starting the third week of treatment. Prolactin concentrations were measured using competitive ELISA. Plasma concentrations of prolactin in non-lactating female guanacos have seasonal changes, with a higher concentration (p < .001) in short days (3.50 ± 2.24 ng/ml) than long days (1.10 ± 0.91 ng/ml). Melatonin treatment significantly decreases (p < .05) plasma concentrations of prolactin on the 21st day after the treatment. These findings are the first report of an endogenous circannual rhythm of plasma prolactin concentration and the action of melatonin treatment on prolactin secretion in this wild camelid.

Kisspeptin induces LH release and ovulation in an induced ovulator†.

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus.

Effect of mating on mRNA and protein expression of beta nerve growth factor and its receptor, TrKA, in the oviduct of llama (Lama glama).

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.

New protocol to separate llama sperm without enzymatic treatment using Androcoll-E™.

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p Ëƒ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies.

Corpus luteum vascularization during the maternal recognition of pregnancy in llamas (Lama glama).

The aim of this study was to characterize corpus luteum vascularization and its association with plasma progesterone concentration in early stages of pregnancy, when maternal recognition of pregnancy is expected to occur. In all animals, both plasma progesterone concentration and corpus luteum vascularization increased from Day 6 to Day 8 post-mating and afterwards in non-pregnant llamas they started to decrease to reach basal levels around Days 12 to 14 post-mating, while in pregnant animals, both variables remained elevated until the end of the study. A lineal positive relationship between corpus luteum vascularization and plasma progesterone concentration was observed in pregnant (r2  = .46, p < .0001) and non-pregnant llamas (r2  = .66, p < .0001). Pregnant animals showed higher plasma progesterone concentration and corpus luteum vascularization than the non-pregnant ones from Day 12 post-mating until the end of the study (p Ë‚ .05 and p Ë‚ .01, respectively). These results suggest that maternal recognition of pregnancy should occur before Day 12 post-mating in order to expand luteal lifespan, maintaining corpus luteum vascularization and progesterone production. Also, the assessment of CL vascularization area could be a useful and non-invasive method for early pregnancy diagnosis due to its association with plasma progesterone concentration.

Calving date and its variability as a potential trait in the breeding objective to account for reproductive seasonality in alpacas.

The low fertility and offspring survival indicators in alpacas can be partially due to their particularity seasonal reproduction that reduces the opportunities of the females to become pregnant within a season, with the survival of the offspring concerned by the availability of food and exposure to diseases that depends on the calving date. Optimizing the date of delivery and reducing its variability are shown as eligible criteria that could be used as selection criteria within the genetic improvement programmes in alpacas, the calving date being a much more appropriate trait to measure and optimize fertility unlike of age at first calving and the calving interval, this due to the reproductive seasonality in camelids. For this study, 6,533 birth date records were taken between 2001 and 2018 of Peruvian alpacas, to estimate the genetic parameters. Models assuming heterogeneity in the residuals were fitted besides classical homogeneous models to address, not only the possibility of forwarding or delaying the calving date, but also the trend to have parturitions in similar dates. The heritability and repeatability ranged from 0.07 to 0.20 for a homogeneity model and from 0.08 to 0.23 for a heterogeneity model, and suggest the possibility of advancing or delaying the calving date. It should be taken into account that the gestation length of camelids makes it difficult to adapt many reproductive traits, and trying to centre the calving date could delay it. It was concluded the feasibility to genetically select the calving date, also in the production of camels and dromedaries, which have the same reproductive characteristics as alpacas. This selection can be combined with other traits. The heterogeneity model was shown to provide a better fit.

The effect of seminal plasma ß-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells.

BACKGROUND: Nerve growth factor (ß-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified ß-Nerve Growth Factor (ß-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. METHODS: Experiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 µg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama ß-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or ß-NGF treatment. RESULTS: Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified ß-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified ß-NGF with respect to the control group. All treatments except ß-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or ß-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified ß-NGF (10 and 20 h). Experiment II: Only ß-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with ß-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or ß-NGF treatment. CONCLUSIONS: Systemic administration of purified ß-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or ß-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

A non-synonymous SNP in exon 3 of the KIT gene is responsible for the classic grey phenotype in alpacas (Vicugna pacos).

The alpaca classic grey phenotype is of particular interest to the industry. Until now, there were only indirect data suggesting that the KIT gene was involved in the classic grey phenotype. All exons of KIT in three black and three classic silvergrey alpacas were sequenced. Five non-synonymous SNPs were observed. There was only one SNP found that was present only in the silvergrey alpacas, and this was also the only SNP predicted to be damaging. This variant results in a change of a glycine (Gly) to an arginine (Arg) at amino acid position 126 (c.376G>A), occurring in the second Ig-like domain of the extracellular domain of KIT. Basic protein modelling predicted that this variant is likely destabilising. Therefore, an additional 488 alpacas were genotyped for this SNP using the tetra-primer amplification refractory mutation system PCR (Tetra-primer ARMS-PCR). All classic grey alpacas were observed to be heterozygous, and 99.3% of non-grey dark base colour alpacas were found to be homozygous for the wildtype allele in this position. These results confirm that the classic grey phenotype in alpacas is the result of a c.376G>A (p.Gly126Arg) SNP in exon 3 of KIT. These data also support the hypothesis that the grey phenotype is autosomal dominant and that the mutation is most likely homozygous lethal.

Characterization and expression analysis of KIT and MITF-M genes in llamas and their relation to white coat color.

The llama (Lama glama) is a fiber-producing species that presents a wide range of coat colors, among which white is one of the most important for the textile industry. However, there is little information about the molecular mechanisms that control the white phenotype in this species. In domestic mammals, a white coat is usually produced by mutations in the KIT proto-oncogene receptor tyrosine kinase (KIT) and microphthalmia-associated transcription factor (MITF) genes. In this work we have sequenced and described the coding regions of KIT and MITF-M, the melanocyte-specific isoform, and the two transcriptional variants MITF-M(-) and MITF-M(+). Moreover, we studied the expression of these genes in the skin of white and colored llamas. Although no variants were revealed to be associated with white coat color, significant differences between phenotypes were observed in the expression levels of KIT and MITF-M. Interestingly, white llamas expressed less MITF-M(+) than did colored ones, which is consistent with a consequent reduction in the synthesis of melanin. Even though our results indicate that downregulation of KIT and MITF-M expression is involved in white phenotype production in llamas, the causative gene of white coat color remains unknown.

A Pilot Study for Estimating the Cardiopulmonary Signals of Diverse Exotic Animals Using a Digital Camera.

Monitoring the cardiopulmonary signal of animals is a challenge for veterinarians in conditions when contact with a conscious animal is inconvenient, difficult, damaging, distressing or dangerous to personnel or the animal subject. In this pilot study, we demonstrate a computer vision-based system and use examples of exotic, untamed species to demonstrate this means to extract the cardiopulmonary signal. Subject animals included the following species: Giant panda (Ailuropoda melanoleuca), African lions (Panthera leo), Sumatran tiger (Panthera tigris sumatrae), koala (Phascolarctos cinereus), red kangaroo (Macropus rufus), alpaca (Vicugna pacos), little blue penguin (Eudyptula minor), Sumatran orangutan (Pongo abelii) and Hamadryas baboon (Papio hamadryas). The study was done without need for restriction, fixation, contact or disruption of the daily routine of the subjects. The pilot system extracts the signal from the abdominal-thoracic region, where cardiopulmonary activity is most likely to be visible using image sequences captured by a digital camera. The results show motion on the body surface of the subjects that is characteristic of cardiopulmonary activity and is likely to be useful to estimate physiological parameters (pulse rate and breathing rate) of animals without any physical contact. The results of the study suggest that a fully controlled study against conventional physiological monitoring equipment is ethically warranted, which may lead to a novel approach to non-contact physiological monitoring and remotely sensed health assessment of animals. The method shows promise for applications in veterinary practice, conservation and game management, animal welfare and zoological and behavioral studies.

Physical characteristics of gastrointestinal content of llama (Lama glama).

Changes in digesta dry matter (DM) and mean digesta particle size (MPS) along the gastrointestinal tract are well known in ruminants, but not in camelids. We collected digesta from the dorsal (d) and ventral (v) first forestomach compartment (C1), the second forestomach compartment (C2), three proximal segments and the subsequent glandular part of the third compartment (C3A-D), the caecum and the faeces twelve llamas (Lama glama). DM analysis indicates the presence of digesta stratification in the C1, the presence of fluid in the C2 to facilitate the sorting function of this compartment, the fluid-absorbing function of the proximal parts of the C3, the secretion of enzymes and digestive acids in the C3D, and the water-resorbing function of the lower intestinal tract. These findings illustrate the functional resemblance between the gastrointestinal tract of camelids and cattle-like ruminants (C1 equivalent to the rumen with stratified contents, C2 to the reticulum, C3A/B/C to the omasum and C3D to the abomasum). MPS analysis revealed a progressive reduction in MPS from the C1 to the distal C3. This gradual transition is different from the clear-cut threshold in ruminants between the reticulum and the omasum and had so far only been described in dromedaries (Camelus dromedarius). These findings indicate that regardless of the convergent property of rumination and resemblance of general mechanisms involved in contents stratification and particle sorting, differences between ruminants and camelids exist that could be interpreted as a more efficient functionality of the ruminant forestomach.

The relationship between gonadotropin releasing hormone and ovulation inducing factor/nerve growth factor receptors in the hypothalamus of the llama.

BACKGROUND: A molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus. METHODS: Mature llamas (n = 4) were euthanized and their hypothalamic tissue was fixed, sectioned, and processed for immunohistochemistry on free-floating sections. Ten equidistant sections per brain were double stained for immunofluorescence detection of TrkA and GnRH, or p75 and GnRH. RESULTS: Cells immunoreactive to TrkA were detected in most hypothalamic areas, but the majority of cells were detected in the diagonal band of Broca (part of the ventral forebrain) and the supraoptic nuclei and periventricular area. The number of cells immunoreactive to p75 was highest in the diagonal band of Broca and lateral preoptic areas and least in more caudal areas of the hypothalamus (p < 0.05) in a pattern similar to that of TrkA. A low proportion of GnRH neurons were immunoreactive to TrkA (2.5% of total GnRH cells), and no co-localization between GnRH and p75 was detected. GnRH neuron fibers were detected only occasionally in proximity to TrkA immunopositive neurons. CONCLUSIONS: Results do not support the hypothesis that the effect of OIF/NGF is driven by a direct interaction with GnRH neurons, but rather provide rationale for the hypothesis that interneurons exist in the hypothalamus that mediate OIF/NGF-induced ovulation.

Follicular fluid stimulates capacitation and acrosome reaction in alpaca sperm (Vicugna pacos).

The follicular fluid exerts an effect on the sperm capacitation of several species; however, these effects vary according to species, both in the sperm motility and in the subsequent acrosome reaction. In this study, the effect of alpaca follicular fluid (aFF) on the motility and acrosome reaction of alpaca spermatozoa was observed, using follicular fluid of three follicle sizes: small (<3 mm), medium (3-6 mm) and large (>6 mm), in a concentration of 30%. Sperm motility at the first hour of incubation with aFF of small follicles was 48.0%, with aFF of medium follicles it was 43.33% and with aFF of large follicles, it was 34.53%, while control averaged 26.00%. At the second hour, control achieved an average of 28.13%, treatment with aFF from small follicles showed an average of 46.53%, with aFF from medium follicles it was 40.00% and with aFF from large follicles it was 35.60%. The acrosome reaction after 4 hours of incubation was 30.06% for control, whereas for aFF of small follicles it was 66.3%, with aFF of medium follicles it was 58.86% and for aFF of large follicles, it was 67.63%. In the case of sperm motility, a significant difference is demonstrated for all treatments in relation to the control at the first hour, whereas only the treatments with aFF of small and medium follicles show a significant difference with respect to the control at the second hour. In the case of the acrosome reaction, all treatments with follicular fluid show a significant difference with respect to the control. It was concluded that alpaca follicular fluid favours sperm capacitation and the acrosome reaction in alpaca spermatozoa.

Sperm kinematic characterization of alpaca (Vicugna pacos L.) during the reproductive season.

Semen analysis is a key factor when determining the fertility ability in males. South American camelids, and in particular the alpaca, have been studied very little when compared with other farm animals. The aim of this work was to perform the kinematic characterization of alpaca spermatozoa collected directly from the deferent duct by using CASA-Mot (Computer Assisted Semen Analysis for Motility) technology. Samples were obtained every three days throughout the reproductive season during two periods and with a break of seven days in the middle. During both periods, the quality of the sample's motility and kinematics increased over the first two days and then subsequently decreased. This pattern was similar in all animals. It was concluded that the introduction of resting times can be useful to improve sperm quality for artificial insemination purposes in natural conditions.

Administration of progesterone BioRelease LA inhibits follicular growth in llamas (Lama glama) regardless of follicle diameter at the start of treatment.

The aims of the study were twofold: first, the comparison of the pharmacokinetics parameters of two doses of Progesterone BioRelease® LA, (BioRelease Technologies, Lexington, KY, USA) one of 300 mg and other of 150 mg and their effects on ovarian dynamics in llamas. Based on the results from the first study, the aim of the second study was to evaluate the effect of the doses of 150 mg of progesterone on follicular activity considering the stage of the largest follicle at the beginning of treatment. The results in Study 1 showed that both doses of the formulation induced plasma progesterone concentrations higher than 1 ng/ml during the first 6 days of treatment in all females, progesterone concentrations steadily decline until Day 5 following by a slowly decrease. The total amount of progesterone released during treatment was higher in Group 300 than in Group 150 (p = 0.045). Mean maximum concentrations were 14.9 ± 2.24 and 14.3 ± 2.16 ng/ml for Group A versus Group B (p = 0.58), and they were registered on Day 1.5 ± 0.22 and 1.7 ± 0.34 days, respectively (p = 0.10). None of the animals of Group A showed progesterone concentration below 1 ng/ml during all studied period. The treatment applied in Study 2 was efficient in inhibiting the ovarian follicular dynamics and to start a superestimulatory treatment. The use of progesterone Biorelease® LA of 150 mg in comparison with the dose of 300 mg could be more effective in the use of synchronization protocols in llamas for AI or prior to the application of an ovarian superstimulatory treatment.

Effect of Foetal Bovine Serum on sperm motility, acrosome reaction and spermatic interaction to zona pellucida in alpacas (Vicugna pacos).

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm-zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.

Determination of Schirmer tear test-1 values in clinically normal alpacas (Vicugña pacos) in North America.

PURPOSE: To determine the normal reference range for Schirmer tear test-1 (STT-1) values in eyes of healthy alpacas (Vicugña pacos). METHODS: Complete ophthalmic examinations were performed on forty healthy alpacas (80 eyes). STT-1 values were obtained in both eyes of all alpacas using a commercial STT strip of a single lot number. Data were analyzed, using a doubly repeated measures ANOVA design, Student's paired t-test, and Pearson correlation test, with P ≤ 0.05 considered significant. RESULTS: The STT-1 values ± standard deviation (SD) were 20.80 ± 4.01 mm/min OD, 20.00 ± 4.13 mm/min OS, and 20.88 ± 4.04 mm/min OU (range 15.50-30.50 mm/min). No significant differences in STT-1 were found between OD and OS. STT-1 was not significantly affected by breed. Schirmer tear test-1 values were significantly increased by 3.45 mm/min for every 10 year increase in age. CONCLUSIONS: This study provides a reference range of STT-1 in the healthy alpaca which can assist veterinarians in diagnosing potential keratoconjunctivitis sicca, tear film abnormalities, as well as ocular surface diseases in alpacas.

FGF21 regulates melanogenesis in alpaca melanocytes via ERK1/2-mediated MITF downregulation.

Fibroblast growth factor 21 (FGF21) is known as a metabolic regulator to regulate the metabolism of glucose and lipids. However, the underlying mechanism of FGF21 on melanin synthesis remains unknown. Therefore, the current study investigates the effect of FGF21 on melanogenesis in alpaca melanocytes. We transfected the FGF21 into alpaca melanocytes, then detected the melanin contents, protein and mRNA levels of pigmentation-related genes in order to determine the melanogenesis-regulating pathway of FGF21. The results showed that FGF21 overexpression suppressed melanogenesis and decreased the expression of the major target genes termed microphthalmia-associated transcription factor (MITF) and its downstream genes, including tyrosinase (TYR) and tyrosinase-related protein 2 (TRP2). However FGF21 increased the expression of phospho-extracellular signal-regulated kinase (p-Erk1/2). In contrast, FGF21-siRNA, a small interference RNA mediating FGF21 silencing, abolished the inhibition of melanogenesis. Altogether, FGF21 may decrease melanogenesis in alpaca melanocytes via ERK activation and subsequent MITF downregulation, which is then followed by the suppression of melanogenic enzymes and melanin production.